Peripheral protein adsorption to lipid-water interfaces: the free area theory.

In fluid monolayers approaching collapse, phospholipids and their complexes with diacylglycerols hinder adsorption to the monolayer of the amphipathic protein, colipase. Herein, a statistical, free-area model, analogous to that used to analyze two-dimensional lipid diffusion, is developed to describe regulation by lipids of the initial rate of protein adsorption from the bulk aqueous phase to the lipid-water interface. It is successfully applied to rate data for colipase adsorption to phospholipid alone and yields realistic values of the two model parameters; the phospholipid excluded area and the critical free surface area required to initiate adsorption. The model is further developed and applied to analyze colipase adsorption rates to mixed monolayers of phospholipid and phospholipid-diacylglycerol complexes. The results are consistent with complexes being stably associated over the physiologically relevant range of lipid packing densities and being randomly distributed with uncomplexed phospholipid molecules. Thus, complexes should form in fluid regions of cellular membranes at sites of diacylglycerol generation. If so, by analogy with the behavior of colipase, increasing diacylglycerol may not trigger translocation of some amphipathic peripheral proteins until its abundance locally exceeds its mole fraction in complexes with membrane phospholipids.

Real-time measurement of solute partitioning to lipid monolayers.

The interaction of a peripheral protein with a lipid-water interface can show a pronounced dependence on the composition and two-dimensional packing density of the lipids that comprise the interface. We report a novel optical method for measuring the adsorption of macromolecules, such as proteins and nucleic acids, and smaller solutes, such as drugs, to lipid monolayers at the gas-liquid interface. Using fluorescence emission from proteins and a small molecule, we demonstrate that the emissions from these solutes when in the aqueous phase and when associated with the monolayer can be temporally separated. Such separation allows measurement of the extent of solute adsorption, spectral characterization of the adsorbed solute, and characterization of lipid organization using adsorption kinetics. The method does not require, but is compatible with, the solute having different spectral properties in the bulk and surface phases. Indeed, if optical signals from adsorbed and soluble solute are the same or their relationship is known, absolute surface excess of adsorbed solute can be calculated without independent calibration. With appropriate instrumental configuration, the method should be adaptable for screening solutes for interaction with planar monolayers having both well-defined composition and adjustable lipid packing density.

Regulation of lipases by lipid-lipid interactions: implications for lipid-mediated signaling in cells.

Lipases are extracellular peripheral proteins that act at the surface of lipid emulsions stabilized, typically, by phospholipids. At a critical composition lipase activity toward substrates in phospholipid monolayers is discontinuously switched on by a small increase in substrate mole fraction. This occurs in part because lipase binding is inhibited by phospholipids. Binding of the lipase cofactor, colipase, is also inhibited by phospholipids. The initial rate of colipase binding increases abruptly at a substrate mole fraction that is approximately half the critical composition for lipase activity and just above that in substrate-phospholipid complexes. Moreover, complex collapse areas show an approximately 1:1 correlation with phospholipid excluded areas determined from an analysis of colipase adsorption rates. Thus, complexes inhibit colipase binding rate. Additionally, the switching of lipase activity likely occurs when uncomplexed substrate becomes the majority species in the interface. Lipase substrates, e.g. diacylglycerols, are typically the same lipids generated in the cytoplasmic surface of the plasma membrane of stimulated cells. As colipase binding is nonspecific and complexes involving lipase substrates form on the basis of lipid-lipid interactions alone, complexes should form in the plasma membrane of stimulated cells and may regulate protein translocation to the membrane.

Lipid lateral organization in fluid interfaces controls the rate of colipase association.

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.

Cholesterol decreases the interfacial elasticity and detergent solubility of sphingomyelins.

The interfacial interactions of cholesterol with sphingomyelins (SMs) containing various homogeneous acyl chains have been investigated by Langmuir film balance approaches. Low in-plane elasticity among the packed lipids was identified as an important physical feature of the cholesterol-sphingomyelin liquid-ordered phase that correlates with detergent resistance, a characteristic property of sphingolipid-sterol rafts. Changes in the in-plane elastic packing, produced by cholesterol, were quantitatively assessed by the surface compressional moduli (C(s)(-1)) of the monolayer isotherms. Of special interest were C(s)(-1) values determined at high surface pressures (>30 mN/m) that mimic the biomembrane situation. To identify structural features that uniquely affect the in-plane elasticity of the sphingomyelin-cholesterol lateral interaction, comparisons were made with phosphatidylcholine (PC)-cholesterol mixtures. Cholesterol markedly decreased the in-plane elasticity of either SM or PC regardless of whether they were fluid or gel phase without cholesterol. The magnitude of the reduction in in-plane elasticity induced by cholesterol was strongly influenced by acyl chain structure and by interfacial functional groups. Liquid-ordered phase formed at lower cholesterol mole fractions when SM's acyl chain was saturated rather than monounsaturated. At similar high cholesterol mole fractions, the in-plane elasticity within SM-cholesterol liquid-ordered phase was significantly lower than that of PC-cholesterol liquid-ordered phase, even when PCs were chain-matched to the SMs. Sphingoid-base functional groups (e.g., amide linkages), which facilitate or strengthen intermolecular hydrogen bonds, appear to be important for forming sphingomyelin-cholesterol, liquid-ordered phases with especially low in-plane elasticity. The combination of structural features that predominates in naturally occurring SMs permits very effective resistance to solubilization by Triton X-100.

Specificity of the lipid-binding domain of apoC-II for the substrates and products of lipolysis.

Functional similarities between colipase and apolipoprotein C-II (apoC-II) in activating lipases suggest that apoC-II may, like colipase, preferentially interact with interfaces containing the substrates and products of lipolysis. To test this hypothesis, the binding of a peptide comprising residues of the cofactor implicated in lipid binding, apolipoprotein C-II(13-56), and, to a lesser extent, apoC-II, to monomolecular lipid films was characterized. The lipids used were a diacylphosphatidylcholine, a diacylglycerol, and a fatty acid. The peptide had an affinity for the argon-buffer interface and for all lipids consistent with a dissociation constant of <10 nM. Changes in surface pressure accompanying peptide binding were comparable to those reported for native apoC-II and indicate peptide miscibility with each of the lipids tested. The capacity of the surfaces to accommodate the peptide decreased with increasing lipid concentration in the interface, indicating competition between lipid and peptide for interfacial occupancy. At a lipid acyl chain density of 470 pmol/cm2, or 35 A2 per acyl chain, a lower limit of peptide adsorption was reached with all lipids. The limiting level of adsorption to phosphatidylcholine was only 1 pmol/cm2 compared with 6;-7 pmol/cm2 for fatty acid and diacylglycerol. Similar results were obtained with apoC-II. The difference in the extent of protein adsorption to lipid classes suggests that the distribution of apoC-II among lipoproteins will depend on their lipid composition and surface pressure.

Interfacial interactions of ceramide with dimyristoylphosphatidylcholine: impact of the N-acyl chain.

The mixing behavior of dimyristoylphosphatidylcholine (DMPC) with either N-palmitoyl-sphingosine (C16:0-ceramide) or N-nervonoyl-sphingosine (C24:1-ceramide) was examined using monomolecular films. While DMPC forms highly elastic liquid-expanded monolayers, both neat C16:0-ceramide and C24:1-ceramide yield stable solid condensed monomolecular films with small areas and low interfacial elasticity. Compression isotherms of mixed C16:0-ceramide/DMPC films exhibit an apparent condensation upon increasing X(cer16:0) at all surface pressures. The average area isobars, coupled with the lack of a liquid-expanded to condensed phase transition as X(cer16:0) is increased, are indicative of immiscibility of the lipids at all surface pressures. In contrast, isobars for C24:1-ceramide/DMPC mixtures show surface pressure-dependent apparent condensation or expansion and surface pressure-area isotherms show a composition and surface pressure-dependent phase transition. This suggests miscibility, albeit non-ideal, of C24:1-ceramide and DMPC in both liquid and condensed surface phases. The above could be verified by fluorescence microscopy of the monolayers and measurements of surface potential, which revealed distinctly different domain morphologies and surface potential values for the DMPC/C16:0- and DMPC/C24:1-ceramide monolayers. Taken together, whereas C16:0-ceramide and DMPC form immiscible pseudo-compounds, C24:1-ceramide and DMPC are partially miscible in both the liquid-expanded and condensed phases, and a composition and lateral pressure-dependent two-phase region is evident between the liquid-expanded and condensed regimes. Our results provide novel understanding of the regulation of membrane properties by ceramides and raise the possibility that ceramides with different acyl groups could serve very different functions in cells, relating to their different physicochemical properties.

Kinetic behavior of the pancreatic lipase-colipase-lipid system.

Pancreatic lipase is a surface-active protein that binds avidly to interfaces comprised of the substrates and products of lipolysis. However, both lipase binding to substrate-containing particles and subsequent interfacial catalysis are inhibited by a number of amphipathic molecules. The most thoroughly studied of these, phosphatidylcholine, is a common constituent of membranes and intestinal lipid contents. Colipase, a surface-active cofactor of lipase, relieves inhibition by phosphatidylcholine in several ways. Through protein-protein interactions, colipase helps anchor lipase to surfaces and stabilizes it in the open conformation. Within the interface, colipase packs more efficiently with substrates and products of lipolysis than with phosphatidylcholine, thereby concentrating these reactants in the vicinity of colipase. This enrichment of lipase substrates and products in the vicinity of colipase enhances lipase-lipid interactions. The result is that colipase facilitates the adsorption of lipase to the interface and, possibly, increases the availability of substrate to the enzyme. Thus, the functional unit in intestinal lipolysis appears to be a lipase-colipase-reactant complex.

Importance of arginines 63 and 423 in modulating the bile salt-dependent and bile salt-independent hydrolytic activities of rat carboxyl ester lipase.

Previous studies using chemical modification approach have shown the importance of arginine residues in bile salt activation of carboxyl ester lipase (CEL) activity. However, the x-ray crystal structure of CEL failed to show the involvement of arginine residues in CEL-bile salt interaction. The current study used a site-specific mutagenesis approach to determine the role of arginine residues 63 and 423 in bile salt-dependent and bile salt-independent hydrolytic activities of rat CEL. Mutations of Arg(63) to Ala(63) (R63A) and Arg(423) to Gly(423) (R423G) resulted in enzymes with increased bile salt-independent hydrolytic activity against lysophosphatidylcholine, having 6.5- and 2-fold higher k(cat) values, respectively, in comparison to wild type CEL. In contrast, the R63A and R423A mutant enzymes displayed 5- and 11-fold decreases in k(cat), in comparison with wild type CEL, for bile salt-dependent cholesteryl ester hydrolysis. Although taurocholate induced similar changes in circular dichroism spectra for wild type, R63A, and R423G proteins, this bile salt was less efficient in protecting the mutant enzymes against thermal inactivation in comparison with control CEL. Lipid binding studies revealed less R63A and R423G mutant CEL were bound to 1,2-diolein monolayer at saturation compared with wild type CEL. These results, along with computer modeling of the CEL protein, indicated that Arg(63) and Arg(423) are not involved directly with monomeric bile salt binding. However, these residues participate in micellar bile salt modulation of CEL enzymatic activity through intramolecular hydrogen bonding with the C-terminal domain. These residues are also important, probably through similar intramolecular hydrogen bond formation, in stabilizing the enzyme in solution and at the lipid-water interface.

Sphingomyelin interfacial behavior: the impact of changing acyl chain composition.

Sphingomyelins (SMs) containing homogeneous acyl chains with 12, 14, 16, 18, 24, or 26 carbons were synthesized and characterized using an automated Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at constant temperatures between 10 degrees C and 30 degrees C. SM containing lauroyl (12:0) acyl chains displayed only liquid-expanded behavior. Increasing the length of the saturated acyl chain (e.g., 14:0, 16:0, or 18:0) resulted in liquid-expanded to condensed two-dimensional phase transitions at many temperatures in the 10-30 degrees C range. Similar behavior was observed for SMs with lignoceroyl (24:0) or (cerotoyl) 26:0 acyl chains, but isotherms showed only condensed behavior at 10 and 15 degrees C. Insights into the physico-mechanical in-plane interactions occurring within the different SM phases and accompanying changes in SM phase state were provided by analyzing the interfacial area compressibility moduli. At similar surface pressures, SM fluid phases were less compressible than those of phosphatidylcholines with similar chain structures. The area per molecule and compressibility of SM condensed phases depended upon the length of the saturated acyl chain and upon spreading temperature. Spreading of SMs with very long saturated acyl chains at temperatures 30-35 degrees below T(m) resulted in condensed films with lower in-plane compressibilities, but consistently larger cross-sectional molecular areas than the condensed phases achieved by spreading at temperatures only 10-20 degrees below T(m). This behavior is discussed in terms of the enhancement of SM lateral aggregation by temperature reduction, a common approach used during domain isolation from biomembranes.

Acyl chain-length asymmetry alters the interfacial elastic interactions of phosphatidylcholines.

Phosphatidylcholines (PCs) with stearoyl (18:0) sn-1 chains and variable-length, saturated sn-2 acyl chains were synthesized and investigated using a Langmuir-type film balance. Surface pressure was monitored as a function of lipid molecular area at various constant temperatures between 10 degrees C and 30 degrees C. Over this temperature range, 18:0-10:0 PC displayed only liquid-expanded behavior. In contrast, di-14:0 PC displayed liquid-expanded behavior at 24 degrees C and 30 degrees C, but two-dimensional phase transitions were evident at 20 degrees C, 15 degrees C, and 10 degrees C. The average molecular area of 18:0-10:0 PC was larger than that of liquid-expanded di-14:0 PC at equivalent surface pressures, and the shapes of their liquid expanded isotherms were somewhat dissimilar. Analysis of the elastic moduli of area compressibility (Cs(-1)) as a function of molecular area revealed shallower slopes in the semilog plots of 18:0-10:0 PC compared to di-14:0 PC. At membrane-like surface pressures (e.g., 30 mN/m), 18:0-10:0 PC was 20-25% more elastic (in an in-plane sense) than di-14:0 PC. Other PCs with varying degrees of chain-length asymmetry (18:0-8:0 PC, 18:0-12:0 PC, 18:0-14:0 PC, 18:0-16:0 PC) were also investigated to determine whether the higher in-plane elasticity of fluid-phase 18:0-10:0 PC is a common feature of PCs with asymmetrical chain lengths. Two-dimensional phase transitions in 18:0-14:0 PC and 18:0-16:0 PC prevented meaningful comparison with other fluid-phase PCs at 30 mN/m. However, the Cs(-1) values for fluid-phase 18:0-8:0 PC and 18:0-12:0 PC were similar to that of 18:0-10:0 PC (85-90 mN/m). These values showed chain-length asymmetrical PCs to have 20-25% greater in-plane elasticity than fluid-phase PCs with mono- or diunsaturated acyl chains.

Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol.

The effect of cholesterol on the interfacial elastic packing interactions of various molecular species of phosphatidylcholines (PCs) has been investigated by using a Langmuir-type film balance and analyzing the elastic area compressibility moduli (Cs(-1)) as a function of average cross-sectional molecular area. Emphasis was on the high surface pressure regions (pi > or = 30 mN/m) which are thought to mimic biomembrane conditions. Increasing levels of cholesterol generally caused the in-plane elasticity of the mixed monolayers to decrease. Yet, the magnitude of the cholesterol-induced changes was markedly dependent upon PC hydrocarbon structure. Among PC species with a saturated sn-1 chain but different sn-2 chain cis unsaturation levels [e.g., myristate (14:0), oleate (18:1delta9(c), linoleate (18:2delta9,12(c), arachidonate (20:4delta5,8,11,14(c), or docosahexenoate (22:6delta4,7,10,13,16,19(c)], the in-plane elasticity moduli of PC species with higher sn-2 unsaturation levels were less affected by high cholesterol mol fractions (e.g., >30 mol %) than were the more saturated PC species. The largest cholesterol-induced decreases in the in-plane elasticity were observed when both chains of PC were saturated (e.g., di-14:0 PC). When both acyl chains were identically unsaturated, the resulting PCs were 20-25% more elastic in the presence of cholesterol than when their sn-1 chains were long and saturated (e.g., palmitate). The mixing of cholesterol with PC was found to diminish the in-plane elasticity of the films beyond what was predicted from the additive behavior of the individual lipid components apportioned by mole and area fraction. Deviations from additivity were greatest for di-14:0 PC and were least for diarachidonoyl PC and didocosahexenoyl PC. In contrast to Cs(-1) analyses, sterol-induced area condensations were relatively unresponsive to subtle structural differences in the PCs at high surface pressures. Cs(-1) versus average area plots also indicated the presence of cholesterol concentration-dependent, low-pressure (<14 mN/m) phase boundaries that became more prominent as PC acyl chain unsaturation increased. Hence, area condensations measured at low surface pressures often do not accurately portray which lipid structural features are important in the lipid-sterol interactions that occur at high membrane-like surface pressures.

Lateral packing of the pancreatic lipase cofactor, colipase, with phosphatidylcholine and substrates.

The interaction of the pancreatic lipase cofactor colipase with a diacylphosphatidylcholine, acylglycerols, and free fatty acid was investigated by monitoring its adsorption to monomolecular lipid films. Surface pressure and colipase surface concentration were measured as a function of the initial lipid concentration and composition. Colipase adsorbs to a level of 28-30 pmol/cm2 to form a close-packed monolayer of protein and interacts strongly with all lipids when the lipid chain:colipase ratio is 3, the triacylglycerol is excluded from the monolayer phase. Phosphatidylcholine, diacylglycerols, and free fatty acid remain in the monolayer phase up to 25 induces higher levels of colipase adsorption than at lower ratios. This suggests the formation of a novel structure involving fatty acid and/or colipase. Phosphatidylcholine also remains in the interface at lipid chain:colipase ratios >3 but shows little additional interaction with colipase. However, fluorescence microscopy suggests that the phosphatidylcholine and colipase are miscible in the interface. The specificity demonstrated in this study suggests that colipase may regulate the type of surfaces to which colipase and, hence, lipase bind and may control the species distribution of substrate to which bound lipase is exposed.

The affinities of procolipase and colipase for interfaces are regulated by lipids.

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.

Lipid structural reorganization induced by the pancreatic lipase cofactor, procolipase.

Pancreatic colipase and its precursor, procolipase, facilitate interfacial lipid hydrolysis catalyzed by pancreatic lipase. To better understand how procolipase functions, its interactions with mixed-lipid monolayers at the argon-buffer interface have been characterized. The lipid mixtures consisted of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and either 1,3-dioleoylglycerol, a model lipase substrate, or 13,16-cis,cis-docosadienoic acid, a model lipase product. Analysis of the lipid composition dependence of procolipase-induced surface pressure increases shows thermodynamically that procolipase interacts strongly and preferentially with the lipase substrate or product. This finding was confirmed by fluorescence measurements of procolipase interaction with pyrene lipid analogs. Analysis of the quantity of procolipase adsorbed to the lipid monolayers shows that interfacial packing obeys a simple, geometric model. The partial molecular areas obtained for procolipase (708 A2) and the phosphatidylcholine (70 A2) agree with their known cross-sectional areas. However, the areas for the fatty acid (14 A2) and diacylglycerol (18 A2) are less than half the expected values, indicating the formation of substrate multilayers. Overall, the results indicate a previously unrecognized role for procolipase, recruiting substrate laterally to its vicinity and, hence, to pancreatic lipase with which procolipase forms a 1:1 interfacial complex. Accompanying this preferential interaction of procolipase with lipase substrates is their rearrangement normal to the interface. These previously unrecognized properties of this lipase cofactor should have relevance for the regulation of other lipases, like lipoprotein lipase, which are regulated by cofactor proteins.

Cholesterol esterase: a cholesterol transfer protein.

Rat pancreatic cholesterol esterase was examined for its ability to effect sterol transfer between small unilamellar vesicle (SUV) preparations. Sterol exchange was determined using SUV composed of palmitoyloleoylphosphatidylcholine/sterol (65:35) with or without 10 mol % phosphatidylserine or phosphatidic acid. This recently developed assay does not require separation of donor and acceptor vesicles (Butko et al., 1992). Cholesterol esterase stimulated cholesterol exchange when SUV contained phosphatidylserine and even more so in the presence of phosphatidic acid. Cholesterol esterase increased the initial rate of sterol transfer between phosphatidic acid-containing SUV by approximately 80%. The enzyme increased sterol exchange by significantly decreasing the half-times of sterol transfer and by significantly increasing the initial rates of sterol exchange. In the absence of negatively charged phospholipids, cholesterol esterase was ineffective at increasing sterol transfer. Monolayer studies showed that negatively charged phospholipids seem to play a key role in cholesterol esterase adsorption to lipid interfaces. Finally, a mutant cholesterol esterase lacking a histidine (435) residue essential for esterasic catalysis was found to be equally capable of increasing sterol transfer and binding to charged monolayers. In summary, cholesterol esterase enhances sterol transfer in SUV containing negatively charged phospholipids, independent of esterasic activity.

Cholesterol's interfacial interactions with sphingomyelins and phosphatidylcholines: hydrocarbon chain structure determines the magnitude of condensation.

Cholesterol's interfacial interaction with different sphingomyelins and phosphatidylcholines has been investigated using a Langmuir film balance. The average molecular area of cholesterol/sphingomyelin (SM) or cholesterol/phosphatidylcholine (PC) mixed monolayers was determined as a function of film composition from the force-area isotherms measured at 24 degrees C. In contrast to previous results [Lund-Katz, S., Laboda, H. M., McLean, L. R., & Phillips, M. C. (1988) Biochemistry 27, 3416-3423], little difference was observed in equimolar cholesterol's "condensing effect" of SMs compared to PCs when their phase state was similar and when their hydrocarbon structural differences were minimized. For PCs, this meant that one acyl chain had to be long and capable of assuming an extended conformation and thus configurationally similar to the long-chain base of SM. This condition facilitated strong van der Waals attractive interactions with cholesterol's planar steroid ring and was satisfied when the sn-1 acyl chain of PC was either myristate or palmitate. Under these conditions, the structural requirements of the sn-2 chain of PC were mitigated. For instance, at equimolar cholesterol, almost no difference was observed in the apparent molecular area condensations of 1-palmitoyl-2-oleoyl-PC and 1-palmitoyl-2-arachidonoyl-PC at surface pressures between 10 and 40 mN/m. In contrast, the apparent molecular area condensations of dioleoyl-PC and diarachidonoyl-PC were substantially reduced under identical experimental conditions. The results are discussed in terms of the relative importance of phospholipid/sphingolipid hydrocarbon and headgroup structure in determining the extent of interaction with cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol's interfacial interactions with galactosylceramides.

Recently, the influence of acyl structure on galactosylceramide's (GalCer) interfacial phase behavior was studied [Ali, S., Smaby, J. M., & Brown, R.E. (1993) Biochemistry 32, 11696-11703]. Here, we show that acyl structure is a key parameter controlling GalCer's ability to interact with cholesterol. Different chain-pure GalCer species containing saturated (24:0, 18:0, or 10:0), or unsaturated (24:1 delta 15, 22:1 delta 13, or 18:2 delta 9, 12) acyl chains were synthesized. After measurement of the force-area behavior of mixed cholesterol/GalCer films at 24 degrees C, the average molecular area and average compressibility were determined as a function of film composition. Cholesterol exerts only a slight condensing effect when the GalCer species are liquid-ordered [liquid-condensed], with maximum condensation occurring near 0.25 mole fraction. However, cholesterol exerts a marked condensing effect on liquid-disordered (liquid-expanded) GalCer species regardless of whether the acyl chain is saturated or unsaturated. Maximum condensation occurs at cholesterol mole fractions between 0.3 and 0.4. We also compared cholesterol's relative condensing effect on liquid-expanded GalCer versus sphingomyelin. Cholesterol's condensation of either bovine brain or egg sphingomyelin is 25-30% greater than that observed with different liquid-expanded GalCer species. Aside from average area behavior, we assessed cholesterol's interfacial interactions with the various sphingolipids by determining the average compressibility as a function of composition. The compressibility of condensed GalCer derivatives changes very little upon addition of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Is lateral phase separation required for fatty acid to stimulate lipases in a phosphatidylcholine interface?

Lipase-catalyzed oxygen exchange between 13,16-cis,cis-docosadienoic acid and water in liquid-expanded monolayers with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine exhibits abrupt, lipid composition-dependent changes in extent and mechanism [e.g., Muderhwa, J. M. and Brockman, H. L. (1992) J. Biol. Chem. 267, 24184-24192]. The critical nature of this transition suggests possible lateral phase separation of the lipids. This has been addressed by substituting for either lipid species one which can exist in more condensed monolayer states. Analysis of phase transition surface pressures as a function of lipid composition shows that each set of fatty acid-phosphatidylcholine mixtures exhibits a finite range of miscibility in liquid-expanded monolayers. These results strongly suggest that 13,16-cis,cis-docosadienoic acid and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine are miscible under the conditions of the oxygen-exchange experiments. Furthermore, to address more directly the relation of lateral lipid phase separation to lipase regulation, oxygen exchange catalyzed by pancreatic carboxylester and triglyceride lipases was studied using mixed monolayers of [18O]2-docosadienoic acid and 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine. These lipids are miscible in the liquid-expanded state at all compositions. The lipid composition dependencies of both the extent and mechanism of lipase-catalyzed oxygen exchange were essentially identical to those obtained earlier. Thus, lateral lipid phase separation is not required for the critical transition in substrate accessibility to lipases. This finding supports a percolation-based model of lipase regulation within a single surface phase and suggests the "topo-temporal" regulation of lipid-mediated signaling in cells.